scholarly journals Next-Generation Sequencing and Proteomics of Cerebrospinal Fluid From COVID-19 Patients With Neurological Manifestations

2021 ◽  
Vol 12 ◽  
Author(s):  
Haijun Wang ◽  
Zili Zhang ◽  
Junfen Zhou ◽  
Shuqing Han ◽  
Zhenyu Kang ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Proteomic Analysis ◽  
Case Series ◽  
Cellular Interaction ◽  
Neurological Manifestations ◽  
Next Generation ◽  
Serum Samples ◽  
Generation Sequencing ◽  
New Onset

The SARS-CoV-2 and its variants are still hitting the world. Ever since the outbreak, neurological involvements as headache, ageusia, and anosmia in COVID-19 patients have been emphasized and reported. But the pathogenesis of these new-onset neurological manifestations in COVID-19 patients is still obscure and controversial. As difficulty always lay in the diagnosis of neurological infection, current reports to validate the presence of SARS-CoV-2 in cerebrospinal fluid (CSF) almost relied on the basic methods and warranted improvement. Here we reported a case series of 8 patients with prominent new-onset neurological manifestations, who were screened out from a patch of 304 COVID-19 confirmed patients. Next-generation sequencing (NGS) and proteomics were conducted in the simultaneously obtained CSF and serum samples of the selected patients, with three non-COVID-19 patients with matched demographic features used as the controls for proteomic analysis. SARS-CoV-2 RNA was detected in the CSF of four COVID-19 patients and was suspicious in the rest four remaining patients by NGS, but was negative in all serum samples. Proteomic analysis revealed that 185 and 59 proteins were differentially expressed in CSF and serum samples, respectively, and that only 20 proteins were shared, indicating that the proteomic changes in CSF were highly specific. Further proteomic annotation highlighted the involvement of complement system, PI3K-Akt signaling pathway, enhanced cellular interaction, and macrophages in the CSF proteomic alterations. This study, equipped with NGS and proteomics, reported a high detection rate of SARS-CoV-2 in the CSF of COVID-19 patients and the proteomic alteration of CSF, which would provide insights into understanding the pathological mechanism of SARS-CoV-2 CNS infection.

scholarly journals 670. Rapid, Non-invasive Detection of Invasive Mycoplasma hominis Infection using the Karius Test, A Next-Generation Sequencing Test for Microbial Cell-free DNA in Plasma

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S390-S390
Author(s):  
Priya Edward ◽  
William V La Via ◽  
Mehreen Arshad ◽  
Kiran Gajurel
Keyword(s):  
Next Generation Sequencing ◽  
Case Series ◽  
Mycoplasma Hominis ◽  
Microbial Cell ◽  
Next Generation ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Hominis Infection ◽  
Generation Sequencing

Abstract Background Mycoplasma hominis is typically associated with genital infections in women and is a rare cause of musculoskeletal infections often in immunocompromised hosts. Diagnosis of invasive Mycoplasma hominis infections are difficult due to challenges in culturing these organisms. Molecular diagnostics require an index of suspicion which may not be present at the time of tissue sampling. Accurate, rapid diagnosis of Mycoplasma hominis infections are important for antibiotic management. Methods Two cases of invasive Mycoplasma hominis infections are presented in which the Karius test (KT) was used to make the diagnosis. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell-free DNA (mcfDNA). After mcfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of > 1400 organisms. Organisms present above a statistical threshold are reported. Case review was performed for clinical correlation. Results A young woman with lupus nephritis status post renal transplant developed persistent fever with progressive multifocal culture-negative osteoarticular infection despite empiric ceftriaxone. An adolescent female presented with an ascending pelvic infection progressing to purulent polymicrobial peritonitis (see table) requiring surgical debridement and cefipime, metronidazole and micafungin therapy; her course was complicated by progressive peritonitis/abscesses. Karius testing detected high-levels of Mycoplasma hominis mcfDNA in both cases – at 3251 molecules/microliter (MPM) in the first case and 3914 MPM in the second case. The normal range of Mycoplasma hominis mcfDNA in a cohort of 684 normal adults is 0 MPM. The patients rapidly improved with atypical coverage with doxycycline and levofloxaxin. Clinical findings in 2 patients with M. hominis infection detected by the Karius Test Conclusion Open-ended, plasma-based NGS for mcfDNA provides a rapid, non-invasive method to diagnose invasive Mycoplasma hominis infection. This case series highlights the potential to diagnose infections caused by fastidious pathogens to better inform antimicrobial therapy and achieve favorable outcomes. Disclosures William V. La Via, MD, Karius (Employee)

scholarly journals Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Imteyaz Ahmad Khan ◽  
Safoora Rashid ◽  
Nidhi Singh ◽  
Sumaira Rashid ◽  
Vishwajeet Singh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Early Stage ◽  
Ductal Adenocarcinoma ◽  
Next Generation ◽  
Serum Samples ◽  
Tissue Samples ◽  
Non Invasive ◽  
Specific Symptoms ◽  
Generation Sequencing

AbstractEarly-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

scholarly journals Development and Optimization of Metagenomic Next-Generation Sequencing Methods for Cerebrospinal Fluid Diagnostics

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Patricia J. Simner ◽  
Heather B. Miller ◽  
Florian P. Breitwieser ◽  
Gabriel Pinilla Monsalve ◽  
Carlos A. Pardo ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Sample Pretreatment ◽  
Standard Of Care ◽  
Nucleic Acid Amplification ◽  
Next Generation ◽  
Bead Beating ◽  
Dna And Rna ◽  
Positive Threshold ◽  
Generation Sequencing

ABSTRACT The purpose of this study was to develop and optimize different processing, extraction, amplification, and sequencing methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) specimens. We applied mNGS to 10 CSF samples with known standard-of-care testing (SoC) results (8 positive and 2 negative). Each sample was subjected to nine different methods by varying the sample processing protocols (supernatant, pellet, neat CSF), sample pretreatment (with or without bead beating), and the requirement of nucleic acid amplification steps using DNA sequencing (DNASeq) (with or without whole-genome amplification [WGA]) and RNA sequencing (RNASeq) methods. Negative extraction controls (NECs) were used for each method variation (4/CSF sample). Host depletion (HD) was performed on a subset of samples. We correctly determined the pathogen in 7 of 8 positive samples by mNGS compared to SoC. The two negative samples were correctly interpreted as negative. The processing protocol applied to neat CSF specimens was found to be the most successful technique for all pathogen types. While bead beating introduced bias, we found it increased the detection yield of certain organism groups. WGA prior to DNASeq was beneficial for defining pathogens at the positive threshold, and a combined DNA and RNA approach yielded results with a higher confidence when detected by both methods. HD was required for detection of a low-level-positive enterovirus sample. We demonstrate that NECs are required for interpretation of these complex results and that it is important to understand the common contaminants introduced during mNGS. Optimizing mNGS requires the use of a combination of techniques to achieve the most sensitive, agnostic approach that nonetheless may be less sensitive than SoC tools.

scholarly journals Diagnosis of Enterococcus faecalis meningitis associated with long-term cerebrospinal fluid rhinorrhoea using metagenomics next-generation sequencing: a case report

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaobo Zhang ◽  
Chao Jiang ◽  
Chaojun Zhou
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Enterococcus Faecalis ◽  
Bone Defect ◽  
Next Generation ◽  
History Of ◽  
Diagnostic Approaches ◽  
Post Traumatic ◽  
Skull Bone Defect ◽  
Generation Sequencing

Abstract Background Enterococcus faecalis (E. faecalis) meningitis is a rare disease, and most of its occurrences are of post-operative origin. Its rapid diagnosis is critical for effective clinical management. Currently, the diagnosis is focused on cerebrospinal fluid (CSF) culture, but this is quite limited. By comparison, metagenomic next-generation sequencing (mNGS) can overcome the deficiencies of conventional diagnostic approaches. To our knowledge, mNGS analysis of the CSF in the diagnosis of E. faecalis meningitis has been not reported. Case presentation We report the case of E. faecalis meningitis in a 70-year-old female patient without a preceding history of head injury or surgery, but with an occult sphenoid sinus bone defect. Enterococcus faecalis meningitis was diagnosed using mNGS of CSF, and she recovered satisfactorily following treatment with appropriate antibiotics and surgical repair of the skull bone defect. Conclusions Non-post-traumatic or post-surgical E. faecalis meningitis can occur in the presence of occult defects in the cranium, and mNGS technology could be helpful in diagnosis in the absence of a positive CSF culture.

scholarly journals Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of External Ventricular and Lumbar Drainage-Associated Ventriculitis and Meningitis

2020 ◽  
Vol 11 ◽  
Author(s):  
Lingye Qian ◽  
Yijun Shi ◽  
Fangqiang Li ◽  
Yufei Wang ◽  
Miao Ma ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Staphylococcus Epidermidis ◽  
Coincidence Rate ◽  
Next Generation ◽  
Lumbar Drainage ◽  
Gram Positive Bacteria ◽  
Neurosurgical Patients ◽  
Generation Sequencing ◽  
Guided Therapy

Metagenomic next-generation sequencing (mNGS) has become a widely used technology that can accurately detect individual pathogens. This prospective study was performed between February 2019 and September 2019 in one of the largest clinical neurosurgery centers in China. The study aimed to evaluate the performance of mNGS on cerebrospinal fluid (CSF) from neurosurgical patients for the diagnosis of external ventricular and lumbar drainage (EVD/LD)-associated ventriculitis and meningitis (VM). We collected CSF specimens from neurosurgical patients with EVD/LD for more than 24 h to perform conventional microbiological studies and mNGS analyses in a pairwise manner. We also investigated the usefulness of mNGS of CSF for the diagnosis of EVD/LD-associated VM. In total, 102 patients were enrolled in this study and divided into three groups, including confirmed VM (cVM) (39), suspected VM (sVM) (49), and non-VM (nVM) (14) groups. Of all the patients, mNGS detected 21 Gram-positive bacteria, 20 Gram-negative bacteria, and five fungi. The three primary bacteria detected were Staphylococcus epidermidis (9), Acinetobacter baumannii (5), and Staphylococcus aureus (3). The mNGS-positive coincidence rate of confirmed EVD/LD-associated VM was 61.54% (24/39), and the negative coincidence rate of the nVM group was 100% (14/14). Of 15 VM pathogens not identified by mNGS in the cVM group, eight were negative with mNGS and seven were inconsistent with the conventional microbiological identification results. In addition, mNGS identified pathogens in 22 cases that were negative using conventional methods; of them, 10 patients received a favorable clinical treatment; thus, showing the benefit of mNGS-guided therapy.

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